Real-world treatment outcome of direct-acting antivirals and patient survival rates in chronic hepatitis C virus infection in Eritrea

Reliable real-world data on direct acting anti-retroviral (DAA) uptake and treatment outcomes are lacking for patients with hepatitis C virus (HCV) in sub-Saharan Africa. This study provides data on HCV DAA-based treatment outcomes, mortality, loss-to-follow up, and associated factors among patients in Eritrea. A multicenter retrospective observational cohort study was conducted in two tertiary hospitals in Asmara, Eritrea. A structured checklist was used to collect data from patient’s cards. Descriptive and inferential statistics used included means (± Standard deviation (SD), medians (Interquartile range (IQR), chi-squire (χ2), Kaplan–Meier estimates, and multivariate Cox proportional hazard models. A total of 238 patients with median age of 59 years (IQR 50–69 years) were enrolled in the study. Out of the 227 patients initiated on treatment, 125 patients had viral load measurements at 12 weeks after end of treatment (EOT) whereas 102 patients had no viral load measurements at 12 weeks EOT. Among the patients with HCV RNA data post-EOT 12, 116 (92.8%) had sustained viral response (SVR). The prevalence of death and loss-to-follow up (LTFU) were (7.5%, 95% CI 1.7–4.1) and 67 (28.1%, 95% CI 22.3–33.9) translating into an incidence of 1.1 (95% CI 0.8–1.5) per 10,000 person days. Independent predictors of LTFU included the enrollment year (2020: aHR = 2.2, 95% CI 1–4.7; p value = 0.04); Hospital (Hospital B: aHR = 2.2, 95% CI 1–4.7; p value = 0.03) and the FIB-4 score (FIB-Score < 1.45: aHR = 3.7, 95% CI 1.2–11.5; p value = 0.02). The SVR rates achieved in this cohort were high. However, high LTFU and high mortality driven largely by late presentation and suboptimal population screening/case finding, were uncovered. These challenges can be addressed by test-and-treat programs that simultaneously prioritize programmatic screening, decentralization of care, and better patient tracking in the HCV care cascade.

Hepatitis C virus (HCV), a predominantly blood-borne hepatotropic RNA virus with ~ 7 genotypes (Gt1-7) and > 67 confirmed subtypes 1 , has emerged as one of the leading causes of mortality and morbidity worldwide 2 .
According to a recent World Health Organization (WHO) global estimate, the number of people with chronic HCV infection is approximately 71 million (95% confidence interval (CI): 62-79 million) people (1%) 3 and only 20% are aware of their condition 4 .In terms of new infections, the data suggest that approximately 1.5 million new infections are registered per year (global incidence: 23.7 per 100,000) 5 .Most of these new infections have been attributed to iatrogenic causes, injection drug use, vertical transmission, body piercings/traditional scarification, and occupational exposure (e.g.needle-stick injuries), among others 6 .
In general, spontaneous clearance of the virus can occur within 6 months in approximately 30% (95% CI 15-45%) of infected persons 7 .However, 70% (95% CI 55-85%) progress to chronic HCV infection that can remain asymptomatic, and therefore unnoticeable, for decades 7 .Chronic hepatitis C (CHC) viremia is associated with multiple hepatic and extra hepatic sequelae which can lead to mortality [8][9][10] .Collectively, these complications were responsible for approximately 580,000 HCV-related deaths in 2017 and substantial impairments in multiple health-related quality of life (HRQL) indices 11,12 .Additional data suggests that unlike, Human Immunodeficiency virus (HIV), or tuberculosis (TB); mortality has trended upward in the last two decades.This upward trend is projected to increase in low-and middle-income countries (LMICs) if testing and subsequent treatments are not scaled-up 13 .
Fortunately, the development of multiple direct-acting antivirals (DAAs) that can achieve up to 95% cure rate with few adverse reactions has revolutionized treatment 2,14 .The main current therapeutic goal for HCV and prevention of liver disease progression is the sustained viral response (SVR) 15,16 .Beyond cure, SVR is associated with several solid clinical endpoints such as reduced likelihood of decompensated cirrhosis 12,17 .Additional public health benefits include better utility values and a reduction in community transmission rates 12 .In 2016, the potential benefit of effective screening programs and prompt treatment prompted the WHO to develop an improved public health action strategy to eliminate HCV infection by 2030 18 .This strategy calls for the diagnosis of 90% of infected individuals and the treatment of 80% of eligible patients.
Emerging evidence suggests that these goals are unlikely to be met 14 .First, some scholars have noted that the prevalence of HCV in LMIC in SSA is poorly researched 3,19 .Of note, the per-capita quantity and quality of real-world studies on DAA effectiveness or data on HCV care cascade performance are extremely rare.In Eritrea, real-world data on therapeutic outcomes is lacking.However, existing data point to low-to-moderate level of HCV infection 20,21 .These circumstances create a strong demand for updated high-quality data on a range of HCV-related issues in Eritrea.Therefore, this study was designed to investigate the treatment outcome of DAA therapy, estimate the frequency loss-to-follow up (LTFU), mortality and incidence rates, and associated factors in two pilot treatment centers in Asmara, Eritrea.

Study design and settings
This observational retrospective cohort study was conducted on patients followed from 2018 to 2021 in the two major chronic HCV care centers in Asmara, Eritrea.See Fig. 1.At present, they serve as the major treatment centers for patients with HCV in Eritrea.Since the inception of the program in 2018, a total of 238 patients have received treatment and follow-up care in the two facilities.Treatment, in general, is guided by the Eritrean Ministry of Health (EMoH) Guideline for Chronic Hepatitis B and C infection (2018).Pan-genotypic DAA regimens recommended in the guideline include Sofosbuvir (SOF) (an NS5B Polymerase Inhibitor-nucleotide analogue), Daclatasvir (DCV) and Velpatasvir (VEL) (NS5A replication complex inhibitors) with SOF/VEL or SOF/DCV for 12 weeks being the preferred combination (See "Supplementary data" Page 2 for clinical laboratory assessments).The HCV viral load count was evaluated using HCV RNA assay.After initiation of DAA, HCV-RNA viral load assays are quantified at 12 and 24 weeks.Of note, these assessments are undertaken at the discretion of the attending physician/clinician and the costs of treatment are covered by the government.

Participants
Patients in the two treatment centers are pooled from the entire country.In general, most patients are referred by clinicians in centers across the country or by transfusion centers following HCV antibody positivity on serological testing.In 2019, several patients were referred following screening campaigns among specific subgroups 22 .All patients aged > 18 years, registered in the two treatment centers were enrolled in this study.See Fig. 2 for additional details.

Data collection tool
Data have been collected via a structured checklist from each patient's clinical card that is routinely documented form for every patient upon enrollment and follow-up.The checklist was structured in a systematic way that would enable data collectors to retrieve data in an orderly fashion and detect systematic errors.

Geographical data and mapping
Zonal boundary data and latitude, and longitude coordinates were obtained from the National Statistical and Geography office in Eritrea.For coordinates that could not be obtained from this office, alternate sources ArcGIS software (ArcMap version 10.7.1 (Esri, Redlands, CA, USA)) and a Google search (See links in supplementary file 1) were employed.

Data processing and analysis
Analysis was conducted using IBM SPSS (version 26) and STATA version 12.0 (STATA Corporation, College Station, TX).Descriptive statistics for categorical variables were analyzed using chi-square (χ 2 )/Fishers exact test and summarized using counts (frequency), proportions (percentages), means (± SD) and medians (interquartile range (IQR).Normality test (Kolmogorov-smirnov test) was conducted prior to any statistical computation and the appropriate parametric (t-test, ANOVA) and nonparametric statistics (Mann-Whitney U and Kruskal Wallis) were used to evaluate differences.Kaplan-Meier curves were used to estimate survival rates and failure rates at different intervals of follow-up.All LTFUs were censored on the date of their last visit.Multivariate Cox regression model was implemented for assessing the variables that predict LTFU.The final results are presented as adjusted hazard ratios (aHR) with a 95% CI.Two-sided p value < 0.05 was considered significant.

Ethical consideration
Ethical approval was obtained from the Ministry of Health research ethics and protocol review committee with a letter of reference (Approval Number: Ref: 01/22).All the information gathered was de-identified, and at most confidentiality was upheld.As the study also included data based on patients' clinical card records, consent for the data access was waived by the ethical committee in place of the patients.All procedures of the study also followed the recommendation of the Declaration of Helsinki Convention.

Factors associated with mortality and LTFU
Table 4 presents factors associated with mortality and LTFU.In this analysis, patients who died had a significantly shorter median duration on treatment with DAA of 31 days (IQR 21-227).In contrast, patients with positive outcomes and LFTU had longer duration of treatment-90 days (IQR 80-144) vs. 84 days (IQR 42-92), respectively (p value = 0.002).Moreover, patients who died had a higher median FIB-4 score, 6.9 (IQR 4.5-6.9)as compared to positive outcome 3.1 (IQR 1.6-6.2) and LTFU 1.3 (IQR: 0.9-6); p value < 0.001.Similarly, mortality was associated with significantly higher median APRI and baseline AST.In contrast, LTFU was higher in both patients who had not started treatment and had been on follow-up for ≤ 12 weeks while Fib-score was relatively lower.

Discussion
Real-world data for treatment programs in SSA is hard to locate in the published literature.Unlike participants in phase 3 randomized controlled trials (RCTs); real-world study cohorts typically include patients with unfavorable conditions.Furthermore, DAAs outcomes can be compromised by clinicians' limited expertise 25 .In this study, all patients had unknown genotypes and unknown fibrosis status.Although consistent details were not available for all patients, the overall SVR rate was 116 (92.8%) for a subset of patients.These results are in line with data from multiple seminal RCT studies (the Phase 3 ASTRAL-1, ASTRAL-2, ASTRAL-3 and ASTRAL-5 trials and POLARIS-3 trials); which reported SVR rates of 93-100% 26 .Even more important, other real world studies and RCTs have similarly shown that DAAs are well tolerated by patients 27 .
Where possible, we computed FIB-4 and APRI scores and evaluated SVR for SOF/VEL and SOF/DCV along cirrhosis strata.In this analysis, our results demonstrated that patients with FIB-4 score < 3.25 had higher SVR (94.5%) compared to patients with FIB-4 score > 3.25(90.5%)for both regimens.Generally, our findings are consistent with the observation that fibrosis may not compromise SOF/VEL and SOF/DCV efficacy, but www.nature.com/scientificreports/decompensated cirrhosis may undermine SVR 28 .To illustrate these points, Abdul and colleagues noted a significant difference in treatment outcomes between patients with disparate Child-Turcotte-Pugh (CTP) classification (95.5% in CTP A vs. 90.8% in CTP B, p value = 0.010) 29 .Literature also suggests that specific HCV genotypes (e.g., HCV-GT4 subtype 4k, 4q, 4p, and 4r) and the emergence of resistance-associated mutations (RAM) 30 may contribute to virological failure.Unfortunately, the possible contribution of these factors to SVR rates in this population is difficult.Therefore, further studies will be required to address this gap.Remarkably, most of the non-responders had high FIB-4 scores, relatively low PLT count (thrombocytopenia can be a surrogate marker of portal hypertension), and high ALT and AST (see "Supplementary data" page 1).Many of these findings align with previous literature which suggested that decompensated liver cirrhosis (CTP B and C), and elevated transaminase levels, among others, are associated with lower SVR 31,32 .Interestingly, posttreatment AST, ALT, FIB-4 score, and APRI score were significantly lower in a sub-set of patients.Furthermore, PLT counts demonstrated limited improvement following treatment.Much of this information concurs partially with literature relating to the clinical benefits of SVR [31][32][33] such as the possible restoration of the liver functional reserve 34 .Altogether it's our conclusion that for most patients, genotype-blind treatment with SOF/VEL or SOF/ DCV regimens is largely satisfactory.Importantly, this outcome reinforces the fact that these regimens can have utility in population-level scale-up measures directed at the elimination of HCV in resource-poor settings in SSA.
Despite the favorable SVR rates, mortality rate was high [18 deaths (7.5% (95% CI 1.7-4.1)]with a significant proportion of deaths occurring in the first 8 weeks after initiation of treatment.The high mortality rate observed in this cohort is probably linked to late presentation of patients.Indeed, patients in this cohort were older (Median (IQR): 60 years (50-69 years) suggesting long-term exposure to HCV.At present, reports suggest that HCV-related cirrhosis can be observed in 5-20% of patients after 20-30 years of chronic infection 35 .Others have noted that cirrhotic patients are at high risk of hepatic decompensation (27.7-39.5% risk over five years) and hepatocellular carcinoma (HCC) (2.8-7.4% in the first year, and 8-16.1% over 5 years) and liver-related mortality 36 .In most countries in SSA, liver transplantation and the cost associated with the management of www.nature.com/scientificreports/patients with End-Stage Liver Disease (ESLD) is prohibitive, therefore, the condition is invariably fatal.This implies that screening of all at-risk populations is a more affordable option for most countries in the region.Therefore, the need for early detection or scale-ups in screening/case-finding along with robust treatment of patients should be prioritized.
In general, experts agree that determination of the severity of liver fibrosis is a challenging but essential component of HCV management 28 .Highlighting this issue, some have noted that limited provider experience; lack of technology for fibrotic staging (e.g.Fibroscan), confirmatory HCV-RNA testing/or genotype determination, as well as clinical chemistry infrastructure, is compromising treatment in LMIC in SSA 37 .Our data corroborate this position.First, the number of hepatologists or specialized internists is severely limited in Eritrea.Clearly, the lack of clinical expertise may compromise hepatic staging-informed care or management of advanced fibrosis/ cirrhosis (an outcome which appears to be common in this setting).In addition, confirmatory HCV-RNA testing is highly centralized and periodic reagent stock-outs have been reported.More importantly, the existing HCV/ HBV standard-of-care guidelines for Eritrea highlight the possible use of APRI, FIB-4 score, and liver elastography for fibrosis determination without specifying the preferred approach.On the latter, we can conclude that the FIB-4 score appears to have a good discriminatory capacity for non-SVR and likelihood of mortality.Overall, better diagnostic performance for FIB-4 score has been reported by multiple investigators 37 .
Lastly, it should be noted that successful completion of treatment is critical for long-term HCV elimination goals.Previous work has shown that LTFU-associated DAA treatment interruptions can be linked to avoidable morbidity and mortality, increased health care costs, preventable HCV transmission, and the development of drug resistance mutations.However, despite overwhelming evidence demonstrating the importance of retention in care; our data demonstrate that LTFU was disproportionately high (67(28.1%,95% CI 22.3-33.9)and that it was the most important non-virological reason for non-attainment of SVR.This outcome stands in stern

Limitations of the study
Although this study addresses an important information gap on HCV literature in SSA, it has some limitations.Firstly, poor documentation of baseline history, laboratory data and post-treatment surveillance data undermined our analyses.Lastly, FIB-4 and APRI scores have not been validated for populations in SSA.

Conclusion
These results provide the first primary data on treatment outcomes in two HCV treatment programs in Eritrea.Important insights included the fact that SOF/VEL and SOF/DCV are highly effective even in settings where genotypes are unknown.Secondly, mortality rates were relatively high, an outcome that was largely associated with the large proportion of patients with cirrhosis.The high incidence of LTFU was surprising given that care was offered for free.Therefore, more work is needed to monitor and understand the factors behind the high proportion of LTFU in this setting.Going forward, more emphasis should be placed on decentralization of care services and better monitoring during and post-EOT.

Figure 4 .
Figure 4. Proportion of loss to follow-up and Mortality in the Consensus HCV care cascade.

Figure 5 .
Figure 5. Kaplan-Meier curves for cumulative survival, LTFU and mortality of chronic HCV patients followed in the two major treatment centers in Eritrea from 2018-to 2021.(A) Overall cumulative proportion of death; (B) Overall cumulative proportion of LTFU; (C) Cumulative proportion of survival by hospital (D) Cumulative mortality curve by FIB 4 score.
a Mann-Whitney U test; b independent samples t-test.Significant values are in [bold].

Table 3 .
Alterations in laboratory parameters in response to DAA among chronic hepatitis-C infected individuals in Eritrea.AST, Aspartate aminotransferase; ALT, Alanine aminotransferase.IQR, interquartile range; VL, Viral load; SD, Standard deviation.a Wilcoxon signed-rank test, b Paired Samples t-test.Significant values are in [bold].

Table 4 .
Demographic characteristics, patient history, and laboratory measurements with treatment and follow-up outcome at inclusion time in the study among Hepatitis-C infected individuals in Eritrea.